U.S. patent application number 12/922850 was filed with the patent office on 2011-01-20 for monoclonal antibodies capable of reacting with a plurality of influenza virus a subtypes.
Invention is credited to Roberto Burioni, Massimo Clementi.
Application Number | 20110014187 12/922850 |
Document ID | / |
Family ID | 40293277 |
Filed Date | 2011-01-20 |
United States Patent
Application |
20110014187 |
Kind Code |
A1 |
Burioni; Roberto ; et
al. |
January 20, 2011 |
MONOCLONAL ANTIBODIES CAPABLE OF REACTING WITH A PLURALITY OF
INFLUENZA VIRUS A SUBTYPES
Abstract
Monoclonal antibodies directed against the influenza A virus are
described, which have the advantageous and unpredicted property of
being able to bind a plurality of subtypes of the influenza A
virus. One preferred embodiment is the antibody designated as
Fab28, which displays a neutralizing activity against a plurality
of subtypes of the influenza A virus. Anti-idiotype antibodies
directed against the monoclonal antibodies of the invention,
immunogenic or vaccine compositions comprising the monoclonal
antibodies of the invention are also described, as well as
therapeutic, prophylactic and diagnostic applications for the
monoclonal antibodies of the invention. The monoclonal antibodies
of the invention can also be used for testing antibody preparations
to be used as vaccines.
Inventors: |
Burioni; Roberto; (Rimini,
IT) ; Clementi; Massimo; (Milano, IT) |
Correspondence
Address: |
Steinfl & Bruno
301 N Lake Ave Ste 810
Pasadena
CA
91101
US
|
Family ID: |
40293277 |
Appl. No.: |
12/922850 |
Filed: |
March 16, 2009 |
PCT Filed: |
March 16, 2009 |
PCT NO: |
PCT/IB09/51068 |
371 Date: |
September 15, 2010 |
Current U.S.
Class: |
424/131.1 ;
424/147.1; 435/320.1; 435/325; 435/7.1; 436/501; 530/387.2;
530/387.3; 530/388.15; 530/388.3 |
Current CPC
Class: |
C07K 2317/34 20130101;
C07K 2317/54 20130101; C12N 15/74 20130101; A61K 47/6841 20170801;
C07K 2317/622 20130101; C07K 16/1018 20130101; A61P 31/16 20180101;
C07K 2317/21 20130101; G01N 33/686 20130101; C07K 2317/56 20130101;
G01N 2469/00 20130101; C07K 16/4216 20130101; C07K 2317/33
20130101; C07K 2317/55 20130101; C07K 2317/24 20130101; C07K
2317/76 20130101 |
Class at
Publication: |
424/131.1 ;
530/388.3; 530/387.3; 530/388.15; 530/387.2; 435/320.1; 424/147.1;
435/325; 435/7.1; 436/501 |
International
Class: |
A61K 39/395 20060101
A61K039/395; C07K 16/10 20060101 C07K016/10; C07K 16/42 20060101
C07K016/42; C12N 15/63 20060101 C12N015/63; A61K 39/42 20060101
A61K039/42; C12N 5/10 20060101 C12N005/10; G01N 33/566 20060101
G01N033/566; A61P 31/16 20060101 A61P031/16 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 17, 2008 |
IT |
TO2008A000204 |
Claims
1-23. (canceled)
24. A monoclonal antibody directed against the influenza A virus
hemagglutinin antigen, wherein the monoclonal antibody is capable
of binding a plurality of subtypes of the influenza A virus, and/or
the monoclonal antibody has a neutralizing activity against a
plurality of subtypes of the influenza A virus.
25. The monoclonal antibody of claim 24, wherein the monoclonal
antibody has a neutralizing activity against a plurality of
subtypes of the influenza A virus, and wherein the monoclonal
antibody is capable of recognizing hemagglutinin from a plurality
of subtypes of the influenza A virus as the antigen.
26. The monoclonal antibody of claim 24, wherein the plurality of
subtypes of the influenza A virus comprises at least one influenza
A virus subtype containing hemagglutinin H1 and one influenza A
virus subtype containing hemagglutinin H3.
27. The monoclonal antibody of claim 24, wherein the monoclonal
antibody comprises at least one heavy chain variable domain and one
light chain variable domain, with the one heavy chain variable
domain having amino acid sequence SEQ ID NO:1 and the one light
chain variable domain having amino acid sequence SEQ ID NO:2.
28. The monoclonal antibody of claim 27, wherein the one heavy
chain variable domain is encoded by nucleotide sequence SEQ ID NO:3
and the one light chain variable domain is encoded by nucleotide
sequence SEQ ID NO:4.
29. The monoclonal antibody according to claim 24, wherein the
monoclonal antibody is capable of binding a hemagglutinin
conformational epitope specifically recognized by a monoclonal
antibody comprising at least one heavy chain variable domain having
amino acid sequence SEQ ID NO:1 and one light chain variable domain
having amino acid sequence SEQ ID NO:2.
30. The monoclonal antibody of claim 24, wherein the antibody is
selected from the group consisting of whole immunoglobulins and
immunoglobulin fragments comprising at least one heavy chain
variable domain and one light chain variable domain.
31. The monoclonal antibody of claim 30, wherein the immunoglobulin
fragments are selected from the group comprising Fab fragments,
Fab' fragments, F(ab').sub.2 fragments, Fv fragments, single chain
antibodies (scFv).
32. The monoclonal antibody of claim 24, wherein the monoclonal
antibody is a human or humanized antibody.
33. An anti-idiotype antibody that is specifically directed against
the idiotype of the monoclonal antibody of claim 24.
34. An isolated polypeptide selected from SEQ ID NO:1 and SEQ ID
NO:2, or fragment thereof at least 8 amino acids in length capable
of binding a plurality of subtypes of the influenza A virus in
vitro.
35. The isolated polypeptide of claim 34, wherein the isolated
polypeptide has a neutralizing activity against a plurality of
subtypes of the influenza A virus.
36. An isolated nucleotide sequence selected from SEQ ID NO:3 and
SEQ ID NO:4.
37. An expression vector comprising the nucleotide sequences of
claim 36, wherein the expression vector comprises nucleotide
sequence SEQ ID NO:3 and/or nucleotide sequence SEQ ID NO:4.
38. A host cell transformed by the expression vector of claim
37.
39. A pharmaceutical composition comprising an effective amount of
at least one anti-idiotype antibody of claim 33 and isolated
polypeptide according claim 34 and a pharmaceutically acceptable
carrier and/or diluent.
40. A prophylactic or therapeutic medicament for an influenza A
virus infection or a pathology directly or indirectly caused by an
influenza A virus infection, the medicament comprising the
monoclonal antibody of claim 24, the anti-idiotype antibody of
claim 33, or the polypeptide of claim 34.
41. A method to for prophylactic or therapeutic treatment of an
influenza A virus infection or a pathology directly or indirectly
caused by an influenza A virus infection in an individual, the
method comprising administering to the individual the monoclonal
antibody of claim 24, the monoclonal antibody of claim 24, the
anti-idiotype antibody of claim 33, or the polypeptide of claim
34.
42. The method of claim 41, wherein the pathology caused by an
influenza A virus infection is the influenza syndrome.
43. An assay method for detecting, in a biological sample from a
patient, the presence of anti-influenza virus antibodies having a
heterosubtype cross-neutralizing property, the method comprising
contacting the said biological sample with a monoclonal antibody
according to claim 24 as a specific assay reagent.
44. A diagnostic kit comprising the monoclonal antibody of claim 24
as a specific reagent, the kit being designed for use in a method
for detecting or quantifying anti-influenza A virus antibodies
having a heterosubtype cross-neutralizing property in a biological
sample obtained from a patient.
45. An assay method for detecting influenza A virus epitopes in an
immunogenic or vaccine composition, the assay method comprising
contacting the composition with a monoclonal antibody of claim 24
as a specific assay reagent; and detecting influenza A virus
epitopes binding the monoclonal antibody, the detected influenza A
virus epitopes capable of eliciting anti-influenza A virus
antibodies having a heterosubtype cross-neutralizing property
against the influenza A virus in an subject to which the
composition is administered.
Description
[0001] The present invention in general falls within the field of
immunology. More specifically, the invention concerns monoclonal
antibodies directed against the HA (hemagglutinin) antigen of the
influenza A virus.
BACKGROUND OF THE INVENTION
[0002] The annual influenza virus epidemics represent an important
cause of morbidity and mortality throughout the world. In the
United States of America it is estimated that more than 200,000
people are hospitalized each year for syndromes connected to
influenza viruses, with about 40,000 deaths more or less directly
related thereto (Thompson et al., JAMA, 2003, 289:179-186). Apart
from these figures we must also consider the cases, in
exponentially higher numbers, of infected subjects that stay at
home for more or less long periods, with inevitable economic
repercussions due to the loss of working days. A recent work
(Molinari et al., Vaccine, 2007, 25: 5086-5096) has estimated the
medical costs directly related to annual epidemics at 10.4 billions
of US dollars per year, to which 16.3 billions of US dollars must
be added for lost earnings due to absence from work. If in the
calculation we consider other items too, such as the monetization
of the economical losses linked to the death of the infected
subjects, the amount rises to the incredible figure of 87.1
billions of US dollars. These economical data linked with the
annual epidemics, together with a dreaded pandemic that could occur
at any moment in the near future due to the appearance of influenza
viruses new to man, explain the considerable interest in the search
for effective strategies to contain the spread of these
viruses.
[0003] Currently, the only available tool for facing the annual
influenza epidemics in some way is an inactivated trivalent vaccine
containing viral isolate antigens that presumably will be
responsible for the epidemic of the next influenza season. This
kind of prediction, based on epidemiological data linked to early
isolations in some sentinel geographic areas, does not always turn
out to be correct. Thus, there is a not at all negligible risk,
which is present year after year, that the trivalent vaccine
developed for a certain influenza season might prove substantially
ineffective.
[0004] In that case, as well as in the case of a new pandemic, the
only available prophylactic/therapeutic aid would be to resort to
the two available classes of antiviral drugs: the M2 protein
inhibitors (amantadine and rimantadine), and the neuraminidase
inhibitors (oseltamivir and zanamivir). However, in this situation
too, a series of problems can be already expected, related both to
the need to administer the antivirals in a very early stage of the
infection, and to the rapid appearance, which has already occurred
however, of resistant viral isolates.
[0005] An alternative effective strategy could be based on
neutralizing antibody preparations directed against critical viral
proteins and capable of recognizing portions of such proteins which
are shared among the different isolates of influenza viruses.
[0006] For better understanding of the potential of an approach
based on the passive administration of antibodies, it is useful to
briefly mention the main structural features of the influenza
viruses. The influenza viruses belong to the Orthomyxoviridae
family and are characterized by the presence of an envelope derived
from infected cell membranes, on which approximately 500 spikes are
present, also referred to as projections. Such projections consist
of trimers and tetramers from two important viral surface proteins:
hemagglutinin (HA) and neuraminidase (NA). An integral membrane
protein (M2) is also found on the envelope surface, which protein
is present in much lower numbers compared to hemagglutinin and
neuraminidase, and also organized in tetramers.
[0007] The influenza virus is further characterized by the
presence, within the core, of a segmented genome comprised of 8
single stranded RNA fragments. Based on the features of some
proteins within the virion (NP and MD, three influenza virus types
are recognizable: type A, type B, and type C.
[0008] Type A and type B viruses are responsible for the annual
epidemics. Instead, type C viruses are responsible for less severe
syndromes.
[0009] Within type A viruses (the only ones responsible for
pandemics and capable of causing the most severe syndromes even
during annual epidemics), different subtypes are also recognizable
based on the antigenic features of hemagglutinin and neuraminidase.
The subtypes that have affected humans in the course of recent
history are subtypes H1N1 and H3N2 (still circulating at present
and contained in vaccine preparations), as well as subtype H2N2, no
longer circulating since 1968 and responsible for the so called
"Asiatic" flu in 1957. Other subtypes have sporadically affected
humans (H9N2, H7N7, and the so dreaded recent H5N1 subtype), but
they have not succeeded in spreading effectively and displacing the
circulating subtypes.
[0010] The role of the surface proteins is essential in the viral
replication cycle. In particular, hemagglutinin is the protein that
allows the virus to recognize the sialic acid present on the
surface of some cells, and to infect them. Instead, neuraminidase
operates at the end of the viral replication cycle, that is during
the release of new virus particles from the infected cells. Its
function is to assist the release of hemagglutinin of the newly
formed virions from the sialic acid present on the surface of the
cell that produced them. The key role played by these two proteins,
as well as their display on the virus surface, explain why they
represent the main target of the immune response, and why they are
susceptible to a high rate of mutation. In fact, the annual
epidemics are caused by viruses that are more or less different
from the ones of the previous years, and therefore are more or less
effectively able to escape the immune response they stimulated. In
other words, the progressive accumulation of point mutations in
hemagglutinin (mostly) and neuraminidase (secondarily) makes the
protective antibodies, produced in the course of previous
epidemics, on the whole progressively ineffective.
[0011] The main protective role within the anti-influenza immune
response is played by the humoral component. Antibodies exert their
protective role primarily interfering with the binding of
hemagglutinin to sialic acid, thereby preventing infection of the
cells. Such a selective pressure determines the high rate of
mutation in hemagglutinin Sequence studies performed on H3
hemagglutinin subtype from 1968 through 1999 have revealed a total
of 101 amino acid mutations (on a total of approximately 560 amino
acids), with an average of about 3.5 mutations per year. 60% of
mutations which occurred in the studied period were retained in the
circulating viruses the following year too, indicative of the
persistence of an immune-mediated selective pressure. 95% of these
mutations were found in the HA1 hemagglutinin subunit, that is the
one directly involved in the binding to sialic acid. Within such a
high variability, however, some unchanged amino acid residues have
been found, indicative of their essential role in the function of
the protein. These hemagglutinin portions represent a potential
target for a cross-neutralizing response towards the different
subtypes of influenza viruses. However, it is predictable that such
regions will not be able to induce an effective antibody response
in most patients, since the fact that such targets hide in
immunosilent areas has certainly represented a very favorable
evolutionary step for the virus.
[0012] In fact, when referring to anti-influenza immunity, three
different types of immunity must be taken into consideration, which
can be well understood in the light of the data reported above:
[0013] HOMOLOGOUS IMMUNITY: related to the individual isolate. This
type of immunity is always seen after an infection or a
vaccination, but it provides a very limited protection against
other isolates. [0014] HOMOSUBTYPE IMMUNITY: related to isolates
belonging to the same subtype. This type of immunity is often seen
after an infection or a vaccination, but is lost when the mutation
rate in hemagglutinin and/or neuraminidase increases considerably.
[0015] HETEROSUBTYPE IMMUNITY: related to isolates belonging to
different subtypes. This type of immunity is extremely rare both in
case of natural infection and in case of vaccination. From the
strategic point of view, it is the most important immunity, as its
presence and stimulation would be equivalent to the resistance to
infection by every type A influenza virus.
[0016] Until a few years ago, it was thought that the heterosubtype
immunity could be achieved just by stimulating effectively cellular
immunity components directed against less mutated viral proteins,
such as for example the NP protein that constitutes its core.
However, recent studies have shown that mice depleted of CD8
lymphocytes are still able to display a heterosubtype immunity, in
contrast with mice depleted of the type B lymphocyte component
(Nguyen H H, J Inf. Dis. 2001, 183: 368-376). An even more recent
study has confirmed this data, highlighting a crucial role of
antibodies, even if not neutralizing, directed precisely against
epitopes that are conserved among the different subtypes
(Rangel-Moreno et al. The J of Immunol, 2008, 180: 454-463).
OBJECT OF THE INVENTION
[0017] On the basis of the data reported above, one object of the
present invention is to provide a monoclonal antibody, preferably
human or humanized, reactive against the different subtypes of the
influenza A virus.
[0018] Another object of the present invention is to provide a
monoclonal antibody, preferably human or humanized, with
neutralizing activity towards multiple subtypes of the influenza A
virus.
[0019] Such an antibody would be an effective means of prevention
when administered to patients at risk. Furthermore, the use of a
human or humanized monoclonal antibody for human patients would
give a further advantage, as the antibody would certainly be well
tolerated.
[0020] Secondly, by constituting a component of the human antibody
response to this virus, the monoclonal antibody with the
above-mentioned features could represent a key factor for the
design of innovative vaccines capable of inducing an extremely more
effective, protective and broad-range immunity, compared to that
induced by the currently used vaccines.
[0021] However, the achievement of monoclonal antibodies with such
properties has so far proved to be extremely difficult.
[0022] The International patent application WO2007/134327, issued
on Nov. 22, 2007, describes Fab fragments capable, in ELISA assays,
of binding to the HA antigen from various isolates of the influenza
A virus, subtype H5. However, this patent application does not
provide an enabling description of antibodies capable of
recognizing isolates belonging to different subtypes of the
influenza A virus, nor does it describe in an enabling way the
attainment of antibodies with actual neutralizing abilities towards
influenza A viruses belonging to different subtypes.
[0023] Therefore, in spite of the fact that a monoclonal antibody
with the ability to recognize and neutralize the different subtypes
of the influenza A virus has been sought for a long time, such a
need has so far remained frustrated.
DESCRIPTION OF THE INVENTION
[0024] The present inventors have surprisingly succeeded in
providing monoclonal antibodies with the above-mentioned desirable
features.
[0025] Thus, a first object of the present invention is a
monoclonal antibody directed against the hemagglutinin antigen of
the influenza A virus, characterized by being able to bind multiple
subtypes of the influenza A virus.
[0026] A second object of the present invention is a monoclonal
antibody directed against the influenza A virus, characterized by
having a neutralizing activity towards multiple subtypes of the
influenza A virus. Preferably, such a neutralizing monoclonal
antibody recognizes influenza A virus hemagglutinin (HA) as the
antigen.
[0027] The monoclonal antibodies of the invention are preferably
human or humanized antibodies.
[0028] The attainment of human monoclonal antibodies according to
the invention and their binding properties are described in detail
in the experimental section that follows.
[0029] The preparation of humanized antibodies is performed by any
per se known methodology, as for example described in Baca et al,
1997 J. Biol. Chem. 272:10678-84 or Carter et al, 1992, Proc. Natl.
Acad. Sci. 89:4285. Such bibliographic references are provided
exclusively for illustration and not limitation. In fact, other
methodologies for the preparation of humanized antibodies are known
in the prior art and can be used within the present invention.
[0030] The attainment of 6 clones (designated as INF4, INF16,
INF28, INF39, INF43 and INF47) capable of producing monoclonal
antibodies in the form of Fab fragments with the in vitro ability
of binding multiple influenza A virus subtypes is specifically
described in the following experimental section.
[0031] The monoclonal antibody produced by clone INF28 (designated
as Fab28) represents one preferred embodiment of the invention, as
the inventors have experimentally proved that this antibody
displays a neutralizing activity towards multiple influenza A virus
subtypes. For the sake of brevity, such an immunological property
will sometimes be referred to herein below as "heterosubtype
cross-neutralizing activity".
[0032] The Fab28 antibody is characterized by a heavy chain
variable domain with the amino acid sequence SEQ ID NO:1 and a
light chain variable domain with the amino acid sequence SEQ ID
NO:2. The nucleotide sequence encoding for the heavy chain variable
domain is SEQ ID NO:3 and the nucleotide sequence encoding for the
light chain variable domain is SEQ ID NO:4.
[0033] In particular, the experimental section describes the
manufacture of the monoclonal antibodies of the invention as Fab
fragments. However, other antibody forms too, and the manufacture
and use thereof are intended to be part of the scope of the
invention. Non-limiting examples are whole immunoglobulins, or
other kinds of antibody fragments, such as for instance
F(ab').sub.2 fragments or antibody fragments smaller than Fabs, or
peptides that have the same immunological properties as those
experimentally demonstrated for the Fab of the invention.
[0034] Single chain antibodies can be constructed according to the
method described in U.S. Pat. No. 4,946,778 by Ladner et al.,
hereby included as reference. Single chain antibodies comprise the
light and heavy chain variable regions linked by a flexible linker.
The antibody fragment designated as single domain antibody is even
smaller than the single chain antibody, as it comprises only one
isolated VH domain. Techniques for obtaining single domain
antibodies having, at least partially, the same binding ability as
the whole antibody, are described in the prior art. Ward, et al.,
in "Binding Activities of a Repertoire of Single Immunoglobulin
Variable Domains Secreted from Escheria coli," Nature 341:644-646,
describes a screening method for obtaining the variable region of
an antibody's heavy chain (VH single domain antibody) with a
sufficient affinity for the target epitope to bind to it in an
isolated form.
[0035] In the description that follows, the term "antibody" will
then be used to refer to all the embodiments mentioned above,
including whole immunoglobulins, Fab fragments or other antibody
fragment types, single chain antibodies, single domain antibodies,
etc.
[0036] The monoclonal antibodies of the invention may be generated
and used in a free form or in a carrier-conjugated form. A carrier
is any molecule or chemical or biological entity capable of
conjugating with an antibody and making it immunogenic or
increasing its immunogenicity. Non-limiting examples of carriers
are proteins such as KLH (keyhole limpet hemocyanin), edestin,
thyroglobulin, albumins as bovine serum albumin (BSA) or human
serum albumin (HSA), erythrocytes such as sheep erythrocytes
(SRBC), tetanus anatoxin, cholera anatoxin, polyamino acids such as
for example poly(D-lysine:D-glutamic acid) and the like. In order
to facilitate the binding of the antibody to the carrier, the
antibody C-terminus or N-terminus may be modified, for example, by
the insertion of additional amino acid residues, for instance one
or more cysteine residues that are able to form disulfide
bridges.
[0037] Because of their properties, which will be shown in detail
in the experimental section that follows, the monoclonal antibodies
of the invention (especially the antibody Fab28) are particularly
suited for use in medical applications, particularly in the
manufacture of a medicament for the broad-range prophylactic or
therapeutic treatment of influenza A virus infections.
[0038] Thus, the use of a monoclonal antibody of the invention,
preferably the antibody Fab28, for the manufacture of a medicament
for the prophylactic or therapeutic treatment of pathologies caused
by influenza A virus infections, such as for instance the influenza
syndrome, is within the scope of the invention.
[0039] In this context too, the expression "Fab28 antibody"
includes not only the Fab fragments but also any other form into
which the antibody can be prepared, for example whole
immunoglobulins, other kinds of antibody fragments, single chain
antibodies, etc.
[0040] As described in detail in the experimental section, the
monoclonal antibodies have been obtained by molecular biology
techniques starting from an EBV-transformed lymphocyte capable of
producing cross-reactive monoclonal antibodies, thus able to
recognize MDCK cell lysates infected with the two reference
isolates as referred to herein below, which belong to different
subtypes of the influenza A virus: H1N1, strain A/PR/8/34 and H3N2,
strain A/PC/1/73. The specific procedures used to generate the
transformed B cell lines from patients' peripheral blood are
described in the experimental section.
[0041] In addition, the procedures used to clone the genes encoding
the heavy and light chain variable portions of the Fab28 antibody
of the invention are described in the experimental section, as well
as the procedures to produce them recombinantly, both as single
peptides and Fab fragments.
[0042] The ability of the monoclonal antibodies of the invention to
react with cell lysates infected with different influenza A virus
subtypes were verified by ELISA and immunofluorescence. In
addition, a neutralizing assay was carried out in order to verify
the in vitro biological activity of the antibodies. In this assay,
the Fab28 antibody showed a heterosubtype cross-neutralizing
activity towards the reference type A viral isolates as indicated
above.
[0043] The obtained data suggest that the antibodies of the
invention, especially antibody Fab28, are extremely effective in
conferring a passive immunity towards the influenza A virus to the
subjects to whom they are administered, and that, accordingly, they
are particularly useful in the broad-range prophylactic or
therapeutic treatment of influenza A virus infections or
pathologies directly or indirectly caused by influenza A virus
infection. One example of such pathologies is the influenza
syndrome.
[0044] In addition, the identification of the hemagglutinin
conformational epitope that Fab28 binds to is described in the
experimental section. Such a conformational epitope lies between
hemagglutinin HA1 region and HA2 region and includes W357 and T358
residues on HA2 region and N336, 1337 and P338 residues on HA1
region. The numbering of the residues is based on the hemagglutinin
sequence from H1N1/A/PR/8/34 isolate in the database BLAST (SEQ ID
NO: 5).
[0045] Thus, a further object of the invention is a pharmaceutical
composition comprising an effective amount of a monoclonal antibody
of the invention as the active ingredient and a pharmaceutically
acceptable carrier and/or diluent. An effective amount is that
which is able to induce a favourable effect in the subject to which
the composition is administered, for example to neutralize the
influenza A virus or interfere with the virus replication.
[0046] In this context, the term "subject" designates any animal
host to which the composition can be administered, including
humans.
[0047] Non-limiting examples of useful pharmaceutically acceptable
carriers or diluents for the pharmaceutical composition of the
invention include stabilizers such as SPGA, carbohydrates (for
example, sorbitol, mannitol, starch, sucrose, glucose, dextran),
proteins such as albumin or casein, protein-containing agents such
as bovine serum or skimmed milk, and buffers (for example phosphate
buffer).
[0048] The monoclonal antibodies of the invention can also be
advantageously used as diagnostic reagents in an in vitro method
for the detection of anti-influenza A virus antibodies with
identical or similar neutralizing properties in a biological sample
previously obtained from a patient (such as for example a serum,
plasma, blood sample or any other suitable biological
material).
[0049] "Anti-influenza A virus antibodies with identical or similar
neutralizing properties" are antibodies that display a
heterosubtype cross-neutralizing activity versus the influenza A
virus. These antibodies may be found in the biological sample from
the patient as a result of a previous exposure to an influenza A
virus, or because the patient had been previously administered a
monoclonal antibody of the invention for therapeutic or
prophylactic or research purposes.
[0050] An assay method for detecting, in a patient's biological
sample, the presence of anti-influenza A virus antibodies having a
heterosubtype cross-neutralizing activity, comprising contacting
the said biological sample with a monoclonal antibody of the
invention, as a specific assay reagent, is thus included in the
scope of the invention.
[0051] The assay can be a qualitative or quantitative one. The
detection or quantification of anti-influenza A virus antibodies
having a heterosubtype cross-neutralizing activity may be carried
out by, for example, a competitive ELISA assay. Thus, a diagnostic
kit comprising a monoclonal antibody according to the invention as
a specific reagent is also within the scope of the invention, the
said kit being particularly designed for the detection or
quantification of anti-influenza A virus antibodies having a
heterosubtype cross-neutralizing activity towards the influenza A
virus in a biological sample derived from a patient.
[0052] Similarly, the monoclonal antibodies of the invention
(especially antibody Fab28) can be used as specific reagents in an
assay method for detecting or quantifying, in a previously prepared
immunogenic or vaccine composition, epitopes capable of evoking, in
the subject to which such a composition has been administered,
anti-influenza A virus antibodies having neutralizing properties
identical or similar to those of the monoclonal antibody of the
invention, that is a heterosubtype cross-neutralizing activity
towards the influenza A virus.
[0053] Such a method is predicted to be useful for the assessment
of any preparation to be used as a vaccine or immunogenic
preparation, as the recognition by the monoclonal antibody of the
invention could be indicative of the presence, in the immunogenic
preparation and/or vaccine, of one or more epitopes capable of
stimulating the production of antibody clones capable of
recognizing an advantageous epitope, such as for example an epitope
capable of eliciting a heterosubtype immunity against the influenza
A virus.
[0054] Finally, the monoclonal antibodies of the invention may be
used for the manufacture of anti-idiotype antibodies according to
methods per se known. Anti-idiotype antibodies are antibodies
specifically directed towards the idiotype of the broad-range
neutralizing antibodies used to prepare them, and as such are able
to mimic the key epitopes they recognize.
[0055] Therefore, anti-idiotype antibodies directed against a
monoclonal antibody of the invention are also included in the scope
of the invention.
[0056] The following experimental section is provided solely by way
of illustration and not limitation and does not intend to restrict
the scope of the invention as defined in the appended claims. The
claims are an integral part of the description.
EXPERIMENTAL SECTION
1. Selection of the Patients
[0057] The patients enrolled in the study were selected so as to
increase the chances of cloning cross-reactive anti-influenza
antibodies, that is antibodies capable of recognizing, and
potentially of neutralizing, influenza virus isolates belonging to
different subtypes. In particular, it is described that some
individuals, despite continuous exposure to the influenza virus
(sometimes for professional reasons, as physicians, pediatricians,
people working in kindergartens and schools), do not contract the
disease. These rare individuals were thought to be less susceptible
to influenza virus infection due to the development, for still
unknown reasons, of an effective heterosubtype immunity. For this
reason they were thought to be the best candidates for the
generation of human monoclonal antibodies. In particular, the
following inclusion criteria were obeyed: [0058] between 25 and 55
years of age; [0059] recent pathological medical history, for the
ten years preceding the study, negative for clinical influenza
syndromes; [0060] antibody titer higher than 1:1000 against virus
isolates, subtypes H1N1 and H3N2 responsible for the annual
epidemics during the five years preceding the study; [0061] high
neutralizing titer (IC50>=1:400) against virus isolates,
subtypes H1N1 and H3N2 responsible for the annual epidemics during
the five years preceding the study; [0062] detectable neutralizing
titer (IC50>=1:20) against two reference subtype A virus
isolates (A/PR/8/34 subtype H1N1; A/PC/1/73 subtype H3N2); [0063]
no prior anti-influenza vaccination; [0064] compliance to receive
anti-influenza vaccination.
[0065] At vaccination, and about 3 weeks post-vaccination,
approximately 20 ml of blood were drawn from each patient into
heparinized test-tubes.
2. Culture of the Reference Virus Isolates
[0066] MDCK (Madin-Darby canine kidney) cells (ATCC.RTM. no.
CCL-34.TM.) propagated in Modified Eagle Medium (MEM) (GIBCO),
supplemented with 10% inactivated fetal bovine serum (FBS)
(treatment at 56.degree. C. for 30 minutes) (EuroClone), 50
.mu.g/ml penicillin, 100 .mu.g/ml streptomycin (GIBCO) and 2 mM
L-glutamine (EuroClone) were used as the cell line. The cells were
incubated at 37.degree. C. in a 5% CO.sub.2 atmosphere and were
passaged at a 1:3 ratio twice a week. For the experiments described
in this patent application, the following influenza virus isolates
were used: H1N1, strain A/PR/8/34 (ATCC.RTM. no. VR-1469.TM.);
H3N2, strain A/PC/1/73 (ATCC.RTM. no. VR-810), and strain B/Lee/40
(ATCC.RTM. no. VR-101). As the culture medium to grow the virus,
MEM supplemented with 1 .mu.g/ml serum-free trypsin (SIGMA) was
used. The virus stocks were obtained from the culture supernatant
as extracellular viruses. In short, after infecting the cells, the
monolayer was observed daily to monitor the appearance of a
cytopathic effect. Generally 4 days after the infection the
supernatant was collected, centrifuged at 1000 RCF (relative
centrifugal force) for 10 minutes to eliminate the cell debris and
filtered with 0.22 .mu.m filters (MILLIPORE). The supernatant was
then aliquoted and stored at -80.degree. C. as cell-free
viruses.
3. Selection of Monoclonal Anti-Influenza Virus Antibodies from
Peripheral Blood B Lymphocytes
[0067] The production of monoclonal antibodies from patients was
carried out by using a trans-formation method via infection of B
lymphocytes with Epstein-Barr virus (EBV), previously described by
Cole et al, 1984 Cancer Research 22:2750-2753. The supernatant from
the different clones obtained was assessed for the presence of
antibodies by ELISA. Clones capable of producing IgG antibodies in
the supernatant that are able to react in the ELISA against the
cell lysates infected with the two reference isolates, subtypes
H1N1 and H3N2, were then selected for a subsequent
characterization. In particular, MDCK cells were infected with the
aforesaid isolates at a high multiplicity of infection. About 48
hours post-infection, the cells were detached from the flask and
washed twice in PBS. The cell pellets were then suspended in 300
.mu.l of lysis solution (100 mM NaCl, 100 mM Tris pH 8 and 0.5%
Triton-X) and stored in ice for 20 minutes. The cell debris were
centrifuged away at 10000 g for 5 minutes and the supernatant was
stored at -20.degree. C. as a protein extract. As for the
preparation of the control antigen, non-infected cells were treated
in the same way. The supernatant protein concentration was
determined in duplicate using the BCA.TM. Protein Assay Kit
(Pierce). Briefly, the sample protein dosage was determined by
referring to a standard curve obtained by a series of
known-concentration dilutions of bovine serum albumin (BSA). The
absorbance of every sample was measured with a spectrophotometer at
a wavelength of 540 nm The lysates so obtained were then used (300
ng per well) to coat an ELISA plate (COSTAR) that was incubated at
4.degree. C. overnight. The following day, the plate was washed
with distilled water and blocked with PBS/1% BSA (Sigma) for 45
minutes at 37.degree. C. Then, 40 .mu.l of supernatant from each
clone were added to each well, which were incubated for 1 hour at
37.degree. C. After 5 washings (WASHER ETI-SYSTEM, DiaSorin) with
PBS/0.5% Tween-20 (Sigma), 40 .mu.l of peroxidase-conjugated
anti-human Fc (1:4000 in PBS/1% BSA, Sigma) were added to each well
and the plate was incubated for 1 hour at 37.degree. C. After 5
more washings with PBS/0.5% Tween-20, 40 .mu.A of TMB peroxidase
substrate (Pierce) were added to each well. Approximately 15
minutes later, the enzymatic activity was blocked by adding 40
.mu.l of H.sub.2SO.sub.4 and the signal was measured with a
spectrophotometer set at 450 nm Special attention was given to the
supernatant of six putative clones capable of producing
cross-reactive antibodies (designated as cINF4, cINF16, cINF28,
cINF39, cINF43 and cINF47, respectively), i.e. capable of
recognizing both cell lysates infected with the strain belonging to
subtype H1N1 and those infected with the strain belonging to
subtype H3N2.
4. Preparation of Fab Fragments from the Cross-Reactive Clones
[0068] The genes encoding for the monovalent Fab chains capable of
reacting with the influenza virus were cloned into a prokaryotic
expression vector. This allows to avoid problems due to instability
of antibody-producing cell clones, to better characterize the
encoding genes from the molecular point of view, in order to have
molecules that are certainly monoclonal at one's disposal, as well
as increased amounts of each individual antibody.
[0069] The messenger RNA (mRNA) was extracted from the cultured
clones and reverse transcribed using an oligo-dT according to
methods per se known. The cDNAs encoding the light chain and the Fd
fragment (i.e. the heavy chain portion present within the Fab
fragment) were then amplified by described methods (CSH press,
Phage display manual, ed. D. R. Burton, p. A1.6). The so obtained
cDNAs were then cloned into an expression vector per se known,
designated as pCb3/CAF (Burioni et al, J. Imm. Meth, 1988). In
short, the gene (amplified DNA) encoding the heavy chain Fd portion
of each Fab was digested with restriction enzymes XhoI and SpeI
(Roche) for 1.5 hours at 37.degree. C., and subsequently inserted
into the vector's cloning site for heavy chains, in turn digested
with the same enzymes. Instead, the light chains (amplified DNA)
were digested with enzymes Sad and XbaI (Roche) and cloned into the
vector similarly digested.
[0070] The so obtained recombinant constructs for each clone were
used to electro-transform E. coli strain XL1 Blue (made competent
by cold washings in glycerol), according to standardized protocols
for the use of 0.2 cm cuvettes (Voltage: 2500 V; Capacitance: 25
.mu.F; Resistance: 200.OMEGA.). In parallel, the DNA sequences of
the light chain variable part and the heavy chain variable part of
the selected clones were analyzed. The sequences are those provided
in the Sequence Listing. The molecular analysis of the mutational
pattern showed a picture ascribable to antigen-induced somatic
mutation processes for each of the clones.
5. ELISA Assessment of the Monoclonal Fabs Obtained by Cloning into
Pcb3/CAF
[0071] At completion of cloning, 40 recombinant bacterial clones
for each monoclonal antibody were analyzed by ELISA using crude
lysates from bacterial cultures obtained by heat shock. In
particular, clones of bacteria transformed with the construct
PCb3/CAF were inoculated into 10 ml of SB medium containing
ampicillin and tetracycline at 50 .mu.g/ml and 10 .mu.g/ml,
respectively, and were grown under shaking at 37.degree. C. until
reaching an O.D.600=1. Subsequently, a specific inducer
(IPTG--isopropyl.beta.-D-thiogalactopyranoside) was added at the
final concentration of 1 mM and the culture was left shaking at
30.degree. C. overnight. The cells were lysed by heat shock (3
freeze/thawing rounds, at -80.degree. C. and 37.degree. C.,
respectively) and then centrifuged to separate the cell debris from
the Fab-containing supernatant. The soluble Fabs obtained were
assayed by ELISA. 96-Well microtiter plates (Nunc) were coated with
lysates from cells infected with the above-mentioned reference
virus isolates. Lysates obtained from uninfected cells were used as
a negative control. The ELISA plates coated with 300 ng of the
lysates obtained as described were then left at 4.degree. C.
overnight. The next day, after removal of the unbound antigen, the
plates were washed 5 times with PBS, and the unspecific binding
sites were blocked with 3% albumin in PBS for 1 hour at 37.degree.
C. After removal of the blocking solution, the supernatants of the
cell cultures treated as described above and containing the soluble
Fabs were added thereto, followed by an incubation step at
37.degree. C. for 2 hours. After 10 washing cycles with PBS/0.05%
Tween 20, 40 .mu.l of a 1:700 dilution of a polyclonal preparation
of radish peroxidase-conjugated goat anti-human Fab immunoglobulins
(Sigma) in PBS/1% BSA was added thereto. After a 1-hour incubation
at 37.degree. C. and a further series of 10 washes, the substrate
(OPD-o-phenylenediamine) was added to the wells. The plates were
then incubated for 30 minutes at room temperature in the dark. The
reaction was quenched with 1N sulfuric acid and the optical density
was assessed by spectrophotometric reading at 450 nm All the
assayed clones displayed reactivity towards the lysates obtained
from the infected cells. One bacterial clone transformed with an
expression vector containing a gene pair encoding the light chain
of a human antibody and the heavy chain Fd fragment was thus
selected for each of the cross-reactive monoclonals. Such bacterial
clones are able to produce human Fabs capable of binding both the
isolate A/PR/8/34 (H1N1) and the isolate A/PC/1/73 (H3N2). These
clones (with the relative gene pairs) were named INF4, INF16,
INF28, INF39, INF43 and INF47.
6. Purification of the Fabs
[0072] The Fabs produced from the above-listed cross-reactive
clones (from here on indifferently referred to as "clones" or
"Fabs") were thus produced through bacteria transformed with the
described expression vector and then immunoaffinity purified with
columns composed of a sepharose resin containing the protein G
(.about.2 mg/ml), to which a polyclonal preparation of goat
antibodies capable of binding human Fabs (PIERCE, Ill.) was
covalently linked. In short, a single colony of each clone was
inoculated into 10 ml of SB medium containing ampicillin and
tetracycline at 50 .mu.g/ml and 10 .mu.g/ml, respectively. The
culture, which was grown overnight at 37.degree. C., was
sub-inoculated into a flask with 500 ml of SB added with the same
concentration of antibiotics as before. The cells, subsequently
induced by 1 mM IPTG, were left shaking overnight at 30.degree. C.
The culture was centrifuged at 5000 rpm for 25 minutes and the
pellet resuspended in PBS was sonicated. A further centrifugation
at 18,000 rpm for 25 minutes was necessary in order to remove the
cell debris. The supernatant was filtered, and then it was slowly
passed through the above-described sepharose column. Thereafter,
the resin was washed with 10 PBS volumes, and finally the bound
Fabs were eluted with an acidic solution (elution
buffer--H.sub.2O/HCl pH 2,2). The various fractions collected were
neutralized with an appropriate solution (1M Tris pH 9) and
concentrated by ultrafiltration (Centricon, Millipore). The purity
of the purified Fabs was assessed by running one aliquot on a 12%
polyacrylamide/sodium dodecyl sulfate gel (SDS-PAGE). Finally,
sequential dilutions of the purified Fabs were assayed by ELISA as
described. Into each plate, preparations of monoclonal Fabs
directed against HCV E2 glycoprotein were included as negative
controls. The results of this experiment confirmed those previously
obtained with the bacterial lysates.
7. Immunofluorescence Assessment of the Monoclonal Fabs Obtained by
Cloning into PCB3/CAF
[0073] In order to confirm the data achieved by ELISA, the
cross-reactive anti-influenza Fabs were also analyzed by an
immunofluorescence assay. Briefly, the cells from the infected
cultures were trypsinized and, after two washes in PBS, counted
under a microscope with a hematocytometer. The cell suspension was
thus used for the preparation of slides by centrifugation in a
cytocentrifuge (Cytospin4, Shandon Southern Products) at 90 g for 3
minutes. The so prepared slides each contained a total of
2.times.10.sup.5 cells. Control slides were prepared similarly with
uninfected cells. The cells were then fixed and permeabilized at
room temperature with a methanol-acetone solution (used at the
temperature of -20.degree. C.) for 10 minutes. After 3 washes in
PBS, the cells were incubated with the different clones (100
.mu.g/ml) for 30 minutes at 37.degree. C. in a humid chamber and
subsequently washed three times in PBS. The cells were then
incubated for 30 minutes at 37.degree. C. in the humid chamber in
the dark with a fluoresceine isothiocyanate-conjugated goat Fab
(Sigma) diluted 1:200 in Evans Blue. The slides were examined under
a fluorescence microscope (Olympus). A commercial mouse monoclonal
(Argene) specific for the M1 influenza virus protein was used as a
positive control. An antibody directed against the E2 glycoprotein
of the hepatitis C virus (e509; Burioni et al, Hepatology, 1998)
was used as a negative control. All the recombinant Fabs showed, by
immunofluorescence, a reactivity that was specific for both the
cells infected with the strain A/PR/8/34 (H1N1) and those infected
with the strain A/PC/1/73 (H3N2). Instead, no fluorescence was seen
in uninfected cells, B type strain-infected cells, or cells
infected with the negative control antibody.
8. Neutralization Assay
[0074] In order to characterize the in vitro biological activity of
the selected clones, neutralization assays were designed for the
three reference virus isolates used in the study. In short, MDCK
cells were seeded into MEM-10% FBS in a 96-well plate
(2.times.10.sup.4 cells/well). Serial dilutions (from 10.sup.-1 to
10.sup.-8) of the virus stocks, obtained as described above, were
prepared in maintenance medium (MEM with 2% FBS). Each dilution was
repeated six times. When the cultured cells were confluent, the
growth medium was removed and 100 .mu.l of each of the virus
dilutions were added to each well. After 1 hour at 37.degree. C.,
the inocula were removed and 200 .mu.l of MEM medium added with 1
.mu.g/ml trypsin were placed into each well. The viral titer,
expressed as TCID.sub.50 (the dose that infects 50% of the cell
culture), was calculated by applying Reed-Muench's formula:
TCID 50 = infectivity > 50 % - 50 % infectivity > 50 % -
infectivity < 50 % .times. dilution factor ##EQU00001##
[0075] In the light of the obtained data, the virus stock was
diluted so as to use a multiplicity of infection (M.O.I.) of
approximately 0.01 (1 virus particle per 100 cells) in the
neutralization experiment. In the actual neutralization assay, MDCK
cells were seeded in a 24-well plate, with each well containing a
sterile slide. The neutralization experiment was performed on
80%-90% confluent cells, i.e. about 48 hours after the seeding
thereof. Dilutions of the purified Fab fragments were then
prepared, so as to attain 2.5 .mu.g/ml, 5 .mu.g/ml, 10 .mu.g/ml and
20 .mu.g/ml final concentrations for each antibody. Corresponding
dilutions of the e509 anti-HCV antibody were prepared as a negative
control. The various Fab concentrations were then incubated with
the same volume of diluted virus stock (M.O.I.: 0.01) for 1 hour at
37.degree. C. 250 .mu.l of the virus-Fab mix were subsequently
added to the wells containing the cells. A positive control for the
infection was achieved by adding the culture medium alone to the
virus stock. The plate was incubated for 1 hour at 37.degree. C. in
order to allow the non-neutralized virus to adsorb. The inoculum
was then removed and the cells were washed twice with PBS. 1.5 ml
of serum-free medium with 1 .mu.g/ml trypsin were added to each
well. After a 6-hour incubation at 37.degree. C., the cell
monolayer was washed with PBS and fixed with a cold
methanol-acetone solution (1:2 ratio, stored at -20.degree. C.) for
10 minutes at room temperature. The fixed cells were washed and
incubated with 250 .mu.l of a commercial monoclonal anti-M1
antibody (Argene) for 30 minutes at 37.degree. C. in a humid
chamber. The cells were washed with PBS and finally incubated with
a fluoresceine-conjugated goat anti-mouse antibody, diluted in
Evans blue, for 30 minutes at 37.degree. C. in a humid chamber in
the dark. After three washes in PBS, the slides were finally
examined under a fluorescence microscope. The Fabs' neutralizing
activity was determined by counting the single positive cells and
calculating the percentage decrease in the number of infected
cells, compared to the positive control infected with the virus
alone. The neutralization assays were carried out in three separate
sessions for each Fab. Particularly, each clone was assayed against
the two different reference type A influenza strains and the
reference type B strain mentioned previously. In each experiment,
the different Fab dilutions were repeated in triplicate, similarly
to what performed for the negative (Fab e509 anti-E2/HCV) and
positive (virus and medium without Fabs) controls of infection.
[0076] Among the six assayed cross-reactive Fabs, the Fab produced
by clone INF28 showed a heterotype cross-neutralizing activity
against the type A virus isolates. Instead, no reduction was
detected with regard to the infecting ability of type B virus used
in the study, confirming the specificity of the neutralizing
activity observed. In particular, the Fab produced by clone INF28
(called Fab 28) showed an IC.sub.50 (the Fab concentration that
inhibits 50% of infection by the virus isolate assayed) below 5
.mu.g/ml in the case of subtype H1N1 and of approximately 10
.mu.g/ml in the case of subtype H3N2, i.e. concentrations that are
easily obtainable by an in vivo administration of the molecules in
question even without taking into account the considerable increase
in the neutralizing biological activity usually observed when Fabs
are converted into the whole immunoglobulin form, one of the
possible pharmaceutical formulations included within the scope of
the invention.
[0077] FIGS. 1 to 3 summarize the results obtained with Fab 28,
produced by clone INF28, in the different neutralization sessions
performed on the various influenza virus isolates used in the
study. Particularly, FIG. 1 is a graph that illustrates the
neutralization percentage of the virus A/PR/8/34 (H1N1) by
different Fab 28 concentrations. The results obtained with the
human e509 anti-HCV Fab are reported as a negative control. FIG. 2
is a graph that illustrates the neutralization percentage of the
virus A/PC/1/73 (H3N2) by different Fab 28 concentrations. The
results obtained with the human e509 anti-HCV Fab are reported as a
negative control. FIG. 3 is a graph that illustrates the
neutralization percentage of the virus B/Lee/40 by different Fab 28
concentrations. The results obtained with the human e509 anti-HCV
Fab are reported as a negative control.
9. Characterization of the Antigen Recognized by Fab 28: Western
Blot on a Lysate from Infected Cells
[0078] 10 .mu.g of a cell lysate infected with strain A/PR/8/34
(H1N1), prepared as described earlier, were run under native
conditions on a 10% polyacrylamide gel. For this purpose, the
samples were run at 100V for 1 hour in a proper refrigerated tank
(BIORAD). Thereafter, the gel was removed from the electrophoresis
apparatus and incubated for 10 minutes in Transfer Buffer (Tris
base 3 g; Glycine 14.41 g, dH.sub.2O 800 ml, Methanol 200 ml) in
order to eliminate any detergent residue. The transfer onto a
nitrocellulose membrane (Hybond-ECL; Amersham Biosciences) was then
carried out overnight at 30V and 90 mA. The membrane was then
blocked for 1 hour with 5% dried milk dissolved in 1.times.PBS and
thereafter washed three times in 1.times.PBS--0.1% Tween. During
each wash, the membrane was left shaking on a swinging platform for
10 minutes. After which, the different Fabs, diluted in PBS with 5%
dried milk, were added at the concentration of 5 .mu.g/ml. Besides
Fab 28, the following controls were added: e509 as a negative
control; commercial mouse anti-HA whole IgG1 (COVANCE); commercial
mouse anti-M1 whole IgG1 (ARGENE); mouse anti-M2 whole IgG1
(ABCAM); human serum diluted 1:200. Each antibody was left shaking
for 1 hour at room temperature. Thereafter, the membrane was washed
again in PBS as described earlier. The same secondary mouse
(1:1000) or human (1:2000) antibodies as described for the ELISA
assay were then added, depending on the source of the antibody to
be detected. For the detection of the signal, a working solution
was prepared by mixing two substrates (SuperSignal.RTM. West Pico
Chemiluminescent Substrate Pierce) in a 1:1 ratio, being
particularly careful not to expose it to sources of light. The
nitrocellulose membrane was incubated for 5 minutes with the
working solution and then removed and mounted in a HyperCassette
(AMERSHAM). This was developed on a Kodak X-ray film in the dark
room after the necessary exposure time. The described assay was
performed in two different sessions, and in each of them the
membrane portion incubated with Fab 28 showed the presence of a
band weighing slightly less than 80 KDa, consistent with the weight
of the immature form of the viral hemagglutinin (HA0). This was
confirmed by the same band being also displayed on the strip
incubated with the anti-hemagglutinin control antibody. An
analogous band, more intense than the others, was also detected in
the membrane portion incubated with human serum. The result of this
experiment shows that the antibody is directed against the
influenza virus hemagglutinin, perfectly consistent with the
neutralization data, since hemagglutinin is known to be the target
of the immune neutralizing antibody response.
10. Neutralization Assay by Plaque Reduction Assay
[0079] Neutralization assays were carried out by using the plaque
assay technique to assess more accurately the neutralizing activity
of Fab 28. Firstly, preparations of virus isolates, subtypes H1N1
and H3N2, were quantified by plaque assay with the following
protocol. MDCK cells were cultured in six-well plates (Costar) in
MEM medium supplemented with penicillin and streptomycin
(pen/strep), and enriched with 10% fetal bovine serum (FBS). After
the cell monolayer had reached 100% confluence, the wells were
washed with PBS and fresh MEM culture medium supplemented with the
same antibiotics (pen/strep) and trypsin (1 .mu.g/ml) was added
thereto. Serial dilutions of the virus stocks were made in the same
wells, and the virus was left to adsorb for 1 hour at 34.degree. C.
under a 5% CO.sub.2 atmosphere. The medium was then aspirated and
two washes with PBS were done. More MEM supplemented with
antibiotics, trypsin (1 .mu.g/ml) and 0.8% agarose was gently added
at a temperature not over 42.degree. C. After infection, the health
condition of the cell monolayer was checked under a phase contrast
light microscope, and the plates were incubated at 34.degree. C.
under a 5% CO.sub.2 atmosphere. 48 hours after infection, the
agarose layer was removed, being very careful not to damage the
cell monolayer. Thereafter, 70% methanol in water, added with
crystal violet (1% w/v), was added to the wells. The plate was
incubated with permeabilizer/dye at room temperature for 5 minutes.
After incubation, the plate was washed with distilled water at room
temperature and left to dry under a laminar flow for 5 minutes.
Finally, the PFU (plaque forming units) number was assessed under
the phase contrast microscope at 4.times. magnitude. Once the virus
titer had been calculated as PFU, the corresponding TCID50 were
calculated, and that same titer was compared to the titer of the
analogous virus stocks by the end-point limiting-dilution
technique.
[0080] The above titration allowed for quantification of the
viruses for the precise assessment of the activity of Fab 28.
Several plates were set up analogously to the above-mentioned
procedure for titration by plaque assay. A neutralization mix was
thus prepared, which comprised the virus (100 TCID50 per well) and
different concentrations of the Fabs that were used (Fab 28 and
control Fab). In particular, the assay was performed by testing
different concentrations of Fabs (20, 10, 5 and 2.5 .mu.g/ml)
against 100 TCID.sub.50 of the diverse influenza virus strains. The
virus/Fab mixtures were then incubated for 1 hour at 34.degree. C.
under a 5% CO.sub.2 atmosphere. After washing the MDCK cells with
PBS, the pre-incubated preparations were transferred into the wells
having a 100% confluent cell monolayer, then were incubated for 1
hour at 34.degree. C. under a 5% CO.sub.2 atmosphere. The assay was
carried out and interpreted as described previously, by comparing
the number of plaques obtained in the presence of Fab 28 with those
obtained in the presence of the same concentration of the control
Fab.
[0081] The assays were performed using the following influenza
isolates belonging to subtypes H1N1 and H3N2:
H1N1:
[0082] A/Malaya/302/54 [0083] A/PR/8/34
H3N2:
[0083] [0084] A/Aichi/68 [0085] A/Victoria/3/75 [0086] A/Port
Chalmers/1/73
[0087] The results confirmed the neutralizing activity of Fab 28
towards the influenza viruses H1N1 A/Malaya/302/54 and A/PR/8/34,
confirming as well IC.sub.50 values below 2.5 .mu.g/ml. A
heterosubtype neutralizing activity was also confirmed against the
influenza viruses H3N2 A/Aichi/68, A/Victoria/3/75 and A/Port
Chalmers/1/73 (IC.sub.50 approximately 20 .mu.g/ml).
11. Identification of the Epitope Recognized by Fab 28
[0088] Several approaches were followed to identify the
hemagglutinin region recognized by Fab 28, the ability of which to
recognize an epitope, though conformational, had already been
showed by previous experiments. Indeed, Fab 28 resulted capable of
recognizing the protein only in Western blot assays performed under
semi-native conditions (0.1% SDS). The same experiments had also
pointed out the ability of Fab in recognizing only the immature
form of the protein (HA0), but not the individual subunits (HA1 and
HA2). Hemagglutination inhibition assays (HAI) had been carried out
in parallel, with both chicken erythrocytes and human erythrocytes.
Despite the remarkable neutralizing activity, Fab 28 proved to have
no HAI activity, suggesting that it did not bind residues
implicated in the binding between hemagglutinin and sialic
acid.
[0089] For better characterization of the epitope, two
complementary strategies were followed: selection of random peptide
sequences, contained in a phage display library, which were able to
bind the Fab 28 monoclonal; and in vitro induction, by selective
pressure through Fab 28, of viral variants (escape mutants) capable
of escaping the antibody's neutralizing activity.
[0090] Selection from the peptide library by the panning technique
allowed for the identification of a number of peptides capable of
specifically binding the Fab 28 idiotype. All the identified
peptides were analyzed in order to generate a consensus sequence.
Such a consensus sequence was then used for an in silico analysis
of a hemagglutinin crystal belonging to subtype H1N1. By this
analysis it was possible to reveal the regions potentially
recognized by Fab 28. One epitope in particular was subjected to
further analysis, in view of its compatibility with the results
found earlier, and with those generated in parallel with the
approach by the escape mutants. The epitope is localized on the
stem region of hemagglutinin, that is in a portion between regions
HA1 and HA2 (data perfectly consistent with the results achieved in
the Western Blot and HAI assays). The residues critical for the
binding which were identified are the following: W357 and T358 on
region HA2; N336; 1337; P338 on region HA1 (the numbering of the
residues refers to the hemagglutinin sequence from the isolate
H1N1/A/PR/8/34 present in the BLAST database) (SEQ ID NO:5).
[0091] The assay by the escape mutants was carried out by serial
infections of MDCK cells with 100 TCID50 of H1N1/A/PR/8/34 virus in
the presence of 10 .mu.g/ml of Fab 28, i.e. a Fab concentration
equivalent to its IC90 against the isolate in question. Only after
numerous passages, it was possible to detect infection of the cells
in the presence of the Fab, indicative of a mutation occurred in
the virus genome. In fact, escape mutants mutated in two residues
of region HA2, I361 and D362, were selected, which are adjacent to
the region identified by the in silico approach, confirming the
hypothesis that this is the region recognized by Fab 28.
Sequence CWU 1
1
51122PRTHomo sapiens 1Leu Glu Glu Ser Gly Gly Gly Val Val Gln Pro
Gly Arg Ser Leu Arg1 5 10 15Leu Ser Cys Ala Ala Ser Gly Phe Pro Phe
Ser Ser Tyr Gly Met His 20 25 30Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp Val Ala Gly Val 35 40 45Ser Tyr Asp Gly Ser Tyr Lys Tyr
Tyr Ala Asp Ser Val Lys Gly Arg 50 55 60Phe Thr Ile Ser Arg Asp Ser
Ser Lys Ser Thr Leu Tyr Leu Gln Met65 70 75 80Asn Ser Leu Arg Pro
Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Pro 85 90 95Ser Ala Ile Phe
Gly Ile Tyr Ile Ile Leu Asn Gly Leu Asp Val Trp 100 105 110Gly Gln
Gly Thr Thr Val Thr Val Ser Ser 115 1202105PRTHomo sapiens 2Glu Leu
Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly Asp Arg1 5 10 15Val
Thr Ile Thr Cys Arg Ala Thr Gln Gly Ile Ser Ser Trp Leu Ala 20 25
30Trp Tyr Gln Gln Lys Pro Gly Lys Pro Pro Lys Leu Leu Ile Phe Gly
35 40 45Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser
Gly 50 55 60Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
Glu Asp65 70 75 80Phe Ala Thr Tyr Phe Cys Gln Gln Ala His Ser Phe
Pro Leu Thr Phe 85 90 95Gly Gly Gly Thr Lys Val Glu Ile Lys 100
1053366DNAHomo sapiens 3ctcgaggagt ctgggggagg cgtggtccag cctgggaggt
ccctgagact ctcctgtgca 60gcctctggat tccccttcag tagttatggc atgcactggg
tccgccaggc tccaggcaag 120gggctggagt gggtggcagg tgtttcatat
gatggaagtt ataaatacta tgcggactcc 180gtcaagggcc gattcaccat
ctccagagac agttccaaga gcactctata tctgcaaatg 240aacagcctga
gacctgagga cacggctgtg tattactgtg cgagaccttc cgcgattttt
300ggaatataca ttattctaaa cggtttggac gtctggggcc aagggaccac
ggtcaccgtc 360tcttca 3664315DNAHomo sapiens 4gagctcacgc agtctccatc
ttccgtgtct gcatctgtag gagacagagt cactatcact 60tgtcgggcga ctcagggtat
tagtagttgg ttagcctggt atcagcagaa accagggaaa 120ccacctaaac
tcctgatttt tggtgcatct agtttgcaaa gtggggtccc atcaaggttc
180agcggcagtg gatctgggac agatttcact ctcaccatca gcagtctaca
gcctgaagat 240tttgcaactt acttttgtca acaggctcac agtttcccgc
tcactttcgg cggcgggacc 300aaggtggaga tcaaa 3155565PRTHomo sapiens
5Met Lys Ala Asn Leu Leu Val Leu Leu Cys Ala Leu Ala Ala Ala Asp1 5
10 15Ala Asp Thr Ile Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Asp
Thr 20 25 30Val Asp Thr Val Leu Glu Lys Asn Val Thr Val Thr His Ser
Val Asn 35 40 45Leu Leu Glu Asp Ser His Asn Gly Lys Leu Cys Arg Leu
Lys Gly Ile 50 55 60Ala Pro Leu Gln Leu Gly Lys Cys Asn Ile Ala Gly
Trp Leu Leu Gly65 70 75 80Asn Pro Glu Cys Asp Pro Leu Leu Pro Val
Arg Ser Trp Ser Tyr Ile 85 90 95Val Glu Thr Pro Asn Ser Glu Asn Gly
Ile Cys Tyr Pro Gly Asp Phe 100 105 110Ile Asp Tyr Glu Glu Leu Arg
Glu Gln Leu Ser Ser Val Ser Ser Phe 115 120 125Glu Arg Phe Glu Ile
Phe Pro Lys Glu Ser Ser Trp Pro Asn His Asn 130 135 140Thr Asn Gly
Val Thr Ala Ala Cys Ser His Glu Gly Lys Ser Ser Phe145 150 155
160Tyr Arg Asn Leu Leu Trp Leu Thr Glu Lys Glu Gly Ser Tyr Pro Lys
165 170 175Leu Lys Asn Ser Tyr Val Asn Lys Lys Gly Lys Glu Val Leu
Val Leu 180 185 190Trp Gly Ile His His Pro Pro Asn Ser Lys Glu Gln
Gln Asn Leu Tyr 195 200 205Gln Asn Glu Asn Ala Tyr Val Ser Val Val
Thr Ser Asn Tyr Asn Arg 210 215 220Arg Phe Thr Pro Glu Ile Ala Glu
Arg Pro Lys Val Arg Asp Gln Ala225 230 235 240Gly Arg Met Asn Tyr
Tyr Trp Thr Leu Leu Lys Pro Gly Asp Thr Ile 245 250 255Ile Phe Glu
Ala Asn Gly Asn Leu Ile Ala Pro Met Tyr Ala Phe Ala 260 265 270Leu
Ser Arg Gly Phe Gly Ser Gly Ile Ile Thr Ser Asn Ala Ser Met 275 280
285His Glu Cys Asn Thr Lys Cys Gln Thr Pro Leu Gly Ala Ile Asn Ser
290 295 300Ser Leu Pro Tyr Gln Asn Ile His Pro Val Thr Ile Gly Glu
Cys Pro305 310 315 320Lys Tyr Val Arg Ser Ala Lys Leu Arg Met Val
Thr Gly Leu Arg Asn 325 330 335Asn Pro Ser Ile Gln Ser Arg Gly Leu
Phe Gly Ala Ile Ala Gly Phe 340 345 350Ile Glu Gly Gly Trp Thr Gly
Met Ile Asp Gly Trp Tyr Gly Tyr His 355 360 365His Gln Asn Glu Gln
Gly Ser Gly Tyr Ala Ala Asp Gln Lys Ser Thr 370 375 380Gln Asn Ala
Ile Asn Gly Ile Thr Asn Lys Val Asn Thr Val Ile Glu385 390 395
400Lys Met Asn Ile Gln Phe Thr Ala Val Gly Lys Glu Phe Asn Lys Leu
405 410 415Glu Lys Arg Met Glu Asn Leu Asn Lys Lys Val Asp Asp Gly
Phe Leu 420 425 430Asp Ile Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu
Leu Glu Asn Glu 435 440 445Arg Thr Leu Asp Phe His Asp Ser Asn Val
Lys Asn Leu Tyr Glu Lys 450 455 460Val Lys Ser Gln Leu Lys Asn Asn
Ala Lys Glu Ile Gly Asn Gly Cys465 470 475 480Phe Glu Phe Tyr His
Lys Cys Asp Asn Glu Cys Met Glu Ser Val Arg 485 490 495Asn Gly Thr
Tyr Asp Tyr Pro Lys Tyr Ser Glu Glu Ser Lys Leu Asn 500 505 510Arg
Glu Lys Val Asp Gly Val Lys Leu Glu Ser Met Gly Ile Tyr Gln 515 520
525Ile Leu Ala Ile Tyr Ser Thr Val Ala Ser Ser Leu Val Leu Leu Val
530 535 540Ser Leu Gly Ala Ile Ser Phe Trp Met Cys Ser Asn Gly Ser
Leu Gln545 550 555 560Cys Arg Ile Cys Ile 565
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